The Tissue Processing and Sequencing Facility (TPSF) is essential in providing support and resources for the Urologic Oncology Branch (UOB) and for our collaborators. The TPSF handles every biospecimen that is generated within the UOB, processes each specimen in order to preserve biomolecules, keeps an accurate inventory of each procurement, and assists in the scientific analysis of select specimens, for the ultimate goal of elucidating biological pathways relating to kidney, prostate, and bladder cancers. The TPSF processes tissue from nearly 100% of UOB surgeries, as well as a subset of biopsies and other procedures. Typically, there are 3 to 8 surgeries and 10 to 20 biopsies per week, resulting in tissue samples procured from over 400 patients per year, including kidney, prostate, and bladder carcinomas, adrenal tumors, uterine leiomyomas, lymph node metastases, and other specimens relating to sporadic and familial urologic cancer syndromes. Tissue is always procured in cooperation with Surgical Pathology, to ensure proper handling and accurate diagnosis. Tissue is snap frozen, preserved in formalin or glutaraldehyde, or processed for biomolecule (DNA, RNA, protein, metabolite) purification and analysis. In addition, DNA, serum, and plasma are regularly prepared from blood samples taken from patients with inherited syndromes. Whole blood or RNA may also be procured and stored from select patients. Over two dozen blood samples may be processed per week. Finally, the core also procures and processes urine, ascites or thoracic fluids, cyst fluids, and other body fluids. Frozen samples are stored in liquid nitrogen or in a -80 degree centigrade freezer. Specimens are assigned a de-identified lab number and entered into a secure database, Labmatrix. Approximately 2,000 tissue and 1,000 blood specimens have been procured and processed within the past year. The entire UOB tissue repository contains in excess of 25,000 tissue samples, and DNA from over 5,000 blood samples and 800 tumors. Most of the samples were collected at the NIH Clinical Center, and full patient histories are incorporated into Labmatrix. A future goal is for all clinical and laboratory findings to be incorporated into Labmatrix to provide an accessible resource for all of our studies from bench to bedside. A key function of the TPSF is to support clinical trials within the Branch. This past year we handled samples from patients in several clinical trials that are open to accrual: Bevacizumab and Erlotinib for patients with metastatic papillary kidney cancer or HLRCC, Vandetanib and Metformin for patients with HLRCC, SDH-RCC, or papillary kidney cancer, a Met kinase inhibitor INC280 for patients with papillary kidney cancer, a HIF2 inhibitor for patients with VHL, PANVAC-BCG vs. BCG for patients with high grade bladder cancer, and Durvalumab and Vicinium for patients with high grade bladder cancer. Blood and/or urine samples are processed at regular intervals, for the purpose of investigating pharmacodynamic and phamacokinetic effects of the drugs, as well as other cancer biomarkers. Many of the tumor samples from kidney and bladder surgeries are procured under sterile conditions to establish new cell cultures and mouse xenografts. We have generated over 300 kidney cancer cell lines, 92 of which have been extensively characterized for cancer gene mutations. Lines have been generated from hereditary kidney cancer syndromes (BHD, SDHB, SDHD, VHL, BAP1, HPRC, and HLRCC) and from rare kidney cancer types (chromophobe, TFE-3 RCC, and medullary RCC) that provide unique reagents. In the last year, about a dozen kidney tumors and a few prostate and bladder tumors have been placed in cell culture and/or in SCID/BEIG or nude mice, with a subset of these growing viably in the short-term and a small number (three this year) that become immortal. These lines are invaluable for studying both the molecular basis of tumorigenesis and prospective therapies. In a recent study, a subset of our lines were subjected to drug screening in a collaboration with NCATS. Cell lines are kept in standard carbon dioxide or low oxygen incubators or stored in liquid nitrogen, and mice are housed in an appropriate on-site facility. Some lines have been extensively characterized by DNA sequencing and copy number of selected genes, array CGH, karyotype, methylation, realtime PCR, Western blotting, oxygen consumption and extracellular acidification rate, and metabolomics. The collection of DNA samples for the detection and characterization of germline disease mutations has been at the heart of the gene discovery process in the UOB. Several dozen blood samples per year are analyzed by DNA sequencing including next-generation whole exome, whole genome, and targeted gene sequencing, comparative genome hybridization (CGH) analysis, fluorescent in-situ hybridization (FISH), and/or other genetic studies. We continue to use these techniques to discover new mutations, deletions, translocations, and amplifications. Both frozen and formalin-fixed tissues from kidney, prostate, and bladder cancers that have been processed by the TPSF have been characterized extensively within the UOB by immunohistochemistry, quantitative PCR, expression microarrays, Northern and Western blotting, immunoprecipitation, and DNA sequencing. In the past year, a few dozen tumor samples from HLRCC and BAP1-related renal cell carcinoma have been subjected to whole exome sequencing to better characterize the molecular landscape of these rare hereditary tumor types. Glutaraldehyde-fixed tissues have been used for electron microscopy, in order to characterize subcellular organelles. Proper handling of our surgical specimens has been an essential factor in assuring the best quality laboratory results. The UOB is also involved in providing aliquots of many of its procured tumor tissues and blood samples to collaborating laboratories. The Branch has long-standing collaborations in which we distribute tissue to other laboratories for cell culture and immunotherapy for kidney cancer patients, analysis of kidney cancer cellular markers, cancer gene mutation analysis, protein and RNA studies of adrenal masses (pheochromocytomas), and molecular epidemiology studies of prostate cancers. We participated in several Cancer Genome Atlas (TCGA) projects, by contributing clear cell, chromophobe and papillary renal tumors as well as prostate tumors. The sizeable biospecimen collection amassed by the UOB over the last 30 years provides an invaluable resource for both basic and clinical research regarding kidney, prostate and bladder cancers.